Substitution of transducin ser202 by asp abolishes G-protein/RGS interaction.
Known RGS proteins stimulate GTPase activity of Gi and Gq family members, but do not interact with Gsalpha and G12alpha. To determine the role of specific Galpha residues for RGS protein recognition, six RGS contact residues of chimeric transducin alpha-subunit (Gtalpha) corresponding to the residues that differ between Gialpha and Gsalpha have been replaced by Gsalpha residues. The ability of human retinal RGS (hRGSr) to bind mutant Gtalpha subunits and accelerate their GTPase activity was investigated. Substitutions Thr178 --> Ser, Ile181 --> Phe, and Lys205 --> Arg of Gtalpha did not alter its interaction with hRGSr. The Lys176 --> Leu mutant had the same affinity for hRGSr as Gtalpha, but the maximal GTPase stimulation by hRGSr was reduced by approximately 2.5-fold. The substitution His209 --> Gln led to a 3-fold decrease in the affinity of hRGSr for the Gtalpha mutant without significantly affecting the maximal GTPase enhancement. The Ser202 --> Asp mutation abolished Gtalpha recognition by hRGSr. A counteracting replacement of Glu129 by Ala in hRGSr did not restore the interaction of hRGSr with the Gtalpha Ser202 --> Asp mutant. Our data suggest that the Ser residue at position 202 of Gtalpha is critical for the specificity of RGS proteins toward Gi and Gq families of G-proteins. Consequently, the corresponding residue, Asp229 of Gsalpha, is likely responsible for the inability of RGS proteins to interact with Gsalpha.
Natochin
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Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Artemyev
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D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D002417:Cattle; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C097755:arginyl-glycyl-serine; D001224:Aspartic Acid; D012694:Serine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D015293:Transducin